Evaluation of urinary neutrophil gelatinase‐associated lipocalin to detect renal tubular damage in dogs with stable myxomatous mitral valve disease

Abstract Background Dogs with myxomatous mitral valve disease (MMVD) can experience progressive renal tubular damage and dysfunction. The prevalence of renal tubular damage is not known in dogs with stable MMVD. Objective To evaluate renal tubular damage in dogs with stable MMVD by evaluation of urinary neutrophil gelatinase‐associated lipocalin (NGAL). Animals Ninety‐eight MMVD dogs grouped according to the American College of Veterinary Internal Medicine (ACVIM) staging (group B1, n = 23; group B2, n = 27; group C + D, n = 48) and 46 healthy dogs. Methods Multicenter prospective observational study. Serum and urine chemistry including NGAL reported as uNGAL concentration (uNGAL) and normalized with urinary creatinine (uNGALC) were compared between MMVD dogs and healthy controls, and among different MMVD ACVIM stages. Results The MMVD dogs had significantly higher uNGAL and uNGALC (1204 pg/mL; range, 30‐39 732 and 1816 pg/mg; range, 22‐127 693, respectively) compared to healthy dogs (584 pg/mL; range, 56‐4072 and 231 pg/mg; range, 15‐2407, respectively; P = .002 and P < .0001, respectively). Both uNGAL and uNGALC increased with the increasing ACVIM stage (P = .001 and P < .001, respectively). Conclusions and Clinical Importance Renal tubular damage is present in dogs with stable MMVD, as measured by increased uNGAL. This tubular damage is subclinical, occurs in all stages of MMVD even in the absence of azotemia, and increases with the severity of MMVD. Reno‐protective approaches to manage MMVD dogs should be explored to slow the progression of renal tubular damage in these patients.

Renal involvement associated with impaired cardiac function secondary to chronic heart diseases, as in the course of MMVD, is known as cardiorenal syndrome (CRS) type II. 3,6,7 When MMVD is severe enough to cause CHF, persistent renal hypoperfusion, chronic congestion of the kidneys and maladaptive neurohormonal changes associated with chronic sympathetic stimulation occur and contribute to progressive renal damage. 3,7 More specifically, 2 main mechanisms of renal damage in chronic heart diseases have been proposed: intermittent acute kidney injury (AKI) episodes or sustained kidney injury occurring simultaneously with progressive reduction of functional kidney mass. 3 Currently, serum creatinine concentration (sCr) is routinely used to monitor renal function in dogs with MMVD, although it is not a sufficient indicator of early renal damage or worsening organ function and cannot discriminate between functional and structural injury. 3 Neutrophil gelatinase-associated lipocalin (NGAL) is a protein belonging to the lipocalin family normally secreted in low amounts by renal tubular epithelial cells and other specific tissues. 8,9 Freely circulating NGAL is filtered through the glomeruli and almost completely reabsorbed in the proximal tubules. 10 Urinary NGAL concentration is very low under normal physiologic conditions. In active renal tubular injury, NGAL synthesis and secretion are increased and reabsorption is decreased, resulting in increased urinary concentrations of this biomarker. 10,11 Therefore, in human and in veterinary medicine, uNGAL represents an early and sensitive biomarker of renal tubular damage. [12][13][14] Specifically, it has been recognized as 1 of the earliest and most strongly induced proteins in both ischemic and nephrotoxic animal models of renal injury, and also has emerged as a promising biomarker for AKI in the clinical setting. [12][13][14][15] Beyond the kidney, NGAL shows potential as a biomarker in cardiovascular disease. 16 Increased concentrations of both serum and uNGAL have been reported in many cardiovascular conditions in people, including both acute and chronic heart diseases leading to CHF. 16 To our knowledge, an increase in serum NGAL associated with the development of renal dysfunction has been documented in dogs with acute (ie, ongoing) CHF. 17 However, no data regarding uNGAL are available in dogs with stable compensated MMVD.
We aimed to (1) determine whether uNGAL differs between dogs with stable MMVD and healthy controls, and (2) assess uNGAL in dogs with MMVD according to the American College of Veterinary Internal Medicine (ACVIM) staging system. Our hypothesis was that uNGAL increases in dogs with MMVD if compared to healthy control dogs, and that increasing uNGAL is detected with increasing MMVD ACVIM stage.

| MATERIAL AND METHODS
Ours was a multicentric prospective, observational case-control study

| Study population
Privately-owned dogs were enrolled at the cardiology services of both centers. All clinical examinations and cardiac ultrasound examinations were performed or reviewed by a board-certified cardiologist (GR or SC) at both centers. Dogs were eligible for inclusion if affected by MMVD at different ACVIM stages, diagnosed, and classified according to the current guidelines. 1 Included dogs were in stable condition (ie, in the case of dogs in stages C and D, subjects had experienced a previous episode of CHF but were free from clinical and radiographic signs of CHF at the time of enrollment). Dogs were grouped according to ACVIM stage as follows: asymptomatic dogs without echocardiographic evidence of left-sided cardiac remodeling were considered to be in stage B1 (group B1); asymptomatic dogs with echocardiographic signs of left-sided cardiac remodeling (ie, left atrial-to-aortic root ratio ≥1.6 and body weight normalized left ventricular internal diameter in diastole ≥1.7) were considered to be in stage B2 (group B2); dogs in which at least 1 episode of CHF had occurred were considered to be in stage C; whereas dogs experiencing relapses of CHF despite regular administration of more than a total daily dosage of 8 mg/kg of furosemide or an equivalent dosage of torasemide (approximately 10% of the dose of furosemide) along with standard doses of the other medications thought to control the cardiac compromise (eg, pimobendan) were considered to be in stage D. 1 Because of the low number of dogs in stage D, dogs in stages C and D were grouped together for statistical purposes (group C + D). A 12-hour fast had to be enforced before the time of sample collection, but water was always available. In contrast, disturbances of cardiac rhythm associated with MMVD did not were not criteria for exclusion. Because urinary tract inflammation can affect uNGAL concentrations, 18 dogs with pyuria on fresh urine sediment examination (>5 white blood cells per high power field) as well as dogs with clinical and laboratory signs of urinary tract infection or inflammation also were excluded. Cardiac treatments routinely used for MMVD, such as diuretics (eg, furosemide, torasemide), pimobendan, angiotensin converting enzyme inhibitors (ACEI; eg, benazepril, enalapril), spironolactone, and antiarrhythmic drugs were allowed.
Healthy dogs (n = 46) were included as controls for comparative purposes and to calculate the reference interval (RI) for uNGAL. These dogs were owned by medical staff or veterinary students attending the VUH. Dogs were considered healthy in the absence of any signs of illness on clinical examination and within the previous 2 months, and in the absence of clinically relevant clinicopathological abnormalities on CBC, serum biochemistry profile, and urinalysis. Dogs had not received any medications within the preceding 2 months before inclusion in the study, except for routine preventive healthcare.

| Clinical and clinicopathological evaluation
Recorded clinical data were signalment, body weight, medical history, physical and echocardiographic examination findings, current medications, and dosage.
Blood was collected by standard venipuncture using blood vacuum collection systems; concurrent fresh urine samples were collected by spontaneous voiding or cystocentesis. Blood and urine specimens were processed according to standard procedures, and evaluated within 1 hour of collection as previously reported. 19 When it was not possible to perform the bio-chemistry analysis within 1 hour, samples were stored at À80 C for up to a maximum storage period of 2 months.
Urinalysis included urine specific gravity (USG) evaluated using a hand-held refractometer (American Optical, Buffalo, NY), dipstick tests (Combur-Test 10 UX, Roche, Switzerland) read by an automated reader (URISYS 1100, Roche, Switzerland) and confirmed by visual inspection, microscopic sediment evaluation performed at low power field (100Â) and high-power field (400Â), and urine chemistry. Urine sediment was obtained after 5-minute centrifugation at 450g. Urine supernatants were immediately analyzed by dipstick examination, and then used for chemical analyses or stored. Urine chemistry was determined using the same automated chemistry analyzer used for serum biochemistry, and included urinary creatinine (uCr), total protein concentrations and urine protein-to-creatinine ratio (UPC).

| Urinary NGAL evaluation
Urinary NGAL was measured using a commercial sandwich ELISA according to the manufacturer's instructions (Dog NGAL ELISA kit, BIO-PORTO Diagnostics, Hellerup, Denmark) and as previously reported. 13 Aliquots of the urine supernatant of dogs with MMVD and of control dogs enrolled in the study were stored at À80 C for up to 2 months until assayed. The assay was validated in our laboratory for dogs following a validation protocol including linearity and intraassay variation, and validation results were similar to those previously reported and consistent with those reported by the manufacturer. 20 Urine samples from healthy dogs were diluted 1:100 whereas for dogs with MMVD an initial dilution of 1:100 was used, followed by 1:300, 1:500, and 1:900 dilutions for samples where analyte concentration could not be determined. The concentration of uNGAL in the samples was determined by measuring the absorbance of the solution at 450 nm using an appropriate plate reader (DV990BV4 spectrophotometer, N.T. Laboratory s.r.l. Calenzano, Italy) and calculating from a standard curve using curvefitting software (GraphPad Prism software, version 6, San Diego, California).Results were expressed as uNGAL concentrations (pg/mL) and as uNGAL-to-uCr ratio (uNGALC; pg/mg).

| Statistical analysis
Data distribution was assessed using the Shapiro-Wilk test. Data were expressed by standard descriptive statistics and presented as mean ± SD or median and range (minimum-maximum) based on normal or nonnormal data distribution. Data obtained in healthy dogs were used to calculate the RI for uNGAL and uNGALC using the Robust method considering a right-sided distribution. The Mann Whitney U test and the Student's t-test were used to compare dogs with MMVD to healthy control dogs. The Kruskal-Wallis test with post hoc comparison (Conover test) was used to compare continuous variables among dogs with MMVD of different ACVIM stage (group B1 vs B2 vs C + D).
Categorical variables were compared among groups using the chi-squared test. Spearman's correlation coefficient was used to assess potential correlations between variables. Results were considered significant if P < .05. Statistical analyses were performed using a commercially available statistical software package (MedCalc Statistical Software version 19.5.1; Ostend, Belgium).
Median body weight was 8.9 kg (range, 2.4-31.9) and mean age was 11 ± 2.7 years. Dogs in group B1 were significantly younger compared to those in groups B2 and C + D (P = .003). Body weight was significantly higher in group B1 vs group C + D (P = .03). Demographic data for enrolled dogs and for the different groups are reported in Table 1.  (Table 1). No other antiarrhythmic drugs were prescribed because no hemodynamically relevant cardiac rhythm disturbances were found in the study population.

| Clinicopathological data
Among enrolled dogs, 28/98 (27%) were azotemic (sCr between 1.41 and 2.76 mg/dL) whereas 8/48 (17%) had UPC >0.5. Dogs included in group C + D had significantly higher sCr concentration vs B1 and B2 (overall P < .001). The UPC was significantly lower in group B1 dogs compared to dogs in both groups B2 and C + D (overall P = .002). Urea concentration was significantly higher in group C + D vs groups B1 and B2 and significantly higher in group B2 vs group B1 (overall P < .001). In addition, dogs in group C + D had significantly lower serum chloride concentration compared to dogs in group B1 and B2 (overall P < .001). No statistical difference was observed between MMVD dogs in different ACVIM stages for leukocyte count and serum CRP concentration (overall P = .47 and P = .21, respectively). Complete clinicopathological results are reported in Table 2.
T A B L E 1 Demographic data and descriptive statistics of the study population: dogs with myxomatous mitral valve disease (MMVD) in different ACVIM stage (group B1, group B2, group C + D), and healthy dogs

| Urinary NGAL evaluation in healthy and MMVD dogs
The RI for uNGAL and uNGALC obtained in healthy dogs was 0-2300 pg/mL and 0-1400 pg/mg, respectively. No correlation was identified between either uNGAL or uNGALC and age in healthy dogs (r = À.02, P = .17; r = À .01, P = .46, respectively  Table 3; Figures 1 and 2). were not significantly correlated with uNGAL (r = .02, P = .08; r = .14, P = .16; r = .14, P = .59, respectively) and uNGALC (r = .15, P = .12; r = .15, P = .14; r = À.07, P = .78, respectively). Moreover, no correlation was found between uNGAL and uNGALC with daily furosemide dosage (r = .005, P = .97; r = .09, P = .53, respectively). Interestingly, another study documented that serum NGAL represents a marker of renal injury in people with CHF even when sCr is within the RI. 22 In humans, serum and uNGAL also correlate with the clinical severity of CHF and, occasionally, with indices of ventricular structural damage and dysfunction. 16,23 In dogs, recent studies determined that serum and uNGAL act as a sensitive and specific biomarkers of AKI and tubular injury, despite being subjected to potential influence during systemic inflammation. 13,24,25 However, as previously mentioned, the literature concerning NGAL as a biomarker of CRS is currently scarce in dogs. A recent study identified higher serum NGAL in dogs with acute CHF caused by MMVD compared to healthy dogs.
Moreover, higher serum NGAL concentrations were noticed upon admission in dogs with CHF that developed worsening of renal function within 7 days of hospitalization compared to those with stable sCr, thus highlighting the potential role of NGAL as an early biomarker of AKI during acute CHF. 17 To our knowledge, tubular damage in dogs with stable MMVD has never been extensively evaluated. A preliminary evaluation of some biomarkers of kidney damage (clusterin, cystatin B, inosine and NGAL) has been reported previously. 3 In that study, the hypothesized mechanisms contributing to increases in those biomarkers were episodes of intermittent AKI or sustained kidney injury occurring simultaneously with a progressive reduction of functional kidney mass. 3 In that same study, despite being cited, uNGAL results were not reported. Based on our findings, progressive and sustained kidney damage could represent the main mechanism behind CRS type II in MMVD dogs with stable disease, even if the role of episodic AKI cannot be completely ruled out.
The normal function of the cardiorenal axis contributes to normal cardiovascular homeostasis. 7  dogs, 20 and uNGAL seems to predict the risk of progressive vs stable CKD in dogs. 33 Both CKD and chronic valvular disease usually occur in elderly patients, and the estimated prevalence of CKD seems higher in dogs with MMVD than in the general canine population. 4  Clinical Pathology when defining RIs (ie, ≥120 subjects). 43 For this reason, further refinement of RIs should be carried out in the future.
In conclusion, our study identified the presence of renal tubular damage in dogs with stable MMVD. This tubular damage was subclinical, and evident even in the initial stages of the disease in dogs not receiving diuretic treatment. This finding emphasizes that MMVD dogs experience functional kidney impairment beyond that related to hemodynamic changes associated with cardiac disease and diuretics.
Increasing uNGAL along with the worsening of heart disease indicates that renal damage during MMVD in dogs might be progressive and potentially involved in renal fibrosis, renal aging, and CKD development in more advanced MMVD stages. The role of reno-protective approaches in the management of dogs with MMVD should be explored in the future because they can potentially slow progression and decrease complications of CRS.